bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2020‒07‒19
thirty-four papers selected by
Ralitsa Radostinova Madsen
University College London Cancer Institute

  1. Genome Biol. 2020 Jul 15. 21(1): 174
      BACKGROUND: Tumors can evolve and adapt to therapeutic pressure by acquiring genetic and epigenetic alterations that may be transient or stable. A precise understanding of how such events contribute to intratumoral heterogeneity, dynamic subpopulations, and overall tumor fitness will require experimental approaches to prospectively label, track, and characterize resistant or otherwise adaptive populations at the single-cell level. In glioblastoma, poor efficacy of receptor tyrosine kinase (RTK) therapies has been alternatively ascribed to genetic heterogeneity or to epigenetic transitions that circumvent signaling blockade.RESULTS: We combine cell lineage barcoding and single-cell transcriptomics to trace the emergence of drug resistance in stem-like glioblastoma cells treated with RTK inhibitors. Whereas a broad variety of barcoded lineages adopt a Notch-dependent persister phenotype that sustains them through early drug exposure, rare subclones acquire genetic changes that enable their rapid outgrowth over time. Single-cell analyses reveal that these genetic subclones gain copy number amplifications of the insulin receptor substrate-1 and substrate-2 (IRS1 or IRS2) loci, which activate insulin and AKT signaling programs. Persister-like cells and genomic amplifications of IRS2 and other loci are evident in primary glioblastomas and may underlie the inefficacy of targeted therapies in this disease.
    CONCLUSIONS: A method for combined lineage tracing and scRNA-seq reveals the interplay between complementary genetic and epigenetic mechanisms of resistance in a heterogeneous glioblastoma tumor model.
    Keywords:  Epigenetic; Genetic; Glioma stem cells; Insulin receptor substrate/IRS; Lineage tracing; Single-cell RNA-seq; Therapy resistance; Tumor heterogeneity
  2. Nature. 2020 Jul 15.
      Approximately 75% of all breast cancers express the oestrogen and/or progesterone receptors. Endocrine therapy is usually effective in these hormone-receptor-positive tumours, but primary and acquired resistance limits its long-term benefit1,2. Here we show that in mouse models of hormone-receptor-positive breast cancer, periodic fasting or a fasting-mimicking diet3-5 enhances the activity of the endocrine therapeutics tamoxifen and fulvestrant by lowering circulating IGF1, insulin and leptin and by inhibiting AKT-mTOR signalling via upregulation of EGR1 and PTEN. When fulvestrant is combined with palbociclib (a cyclin-dependent kinase 4/6 inhibitor), adding periodic cycles of a fasting-mimicking diet promotes long-lasting tumour regression and reverts acquired resistance to drug treatment. Moreover, both fasting and a fasting-mimicking diet prevent tamoxifen-induced endometrial hyperplasia. In patients with hormone-receptor-positive breast cancer receiving oestrogen therapy, cycles of a fasting-mimicking diet cause metabolic changes analogous to those observed in mice, including reduced levels of insulin, leptin and IGF1, with the last two remaining low for extended periods. In mice, these long-lasting effects are associated with long-term anti-cancer activity. These results support further clinical studies of a fasting-mimicking diet as an adjuvant to oestrogen therapy in hormone-receptor-positive breast cancer.
  3. Am J Respir Cell Mol Biol. 2020 Jul 15.
      Idiopathic pulmonary fibrosis is a fatal disease characterized by the TGF-β-dependent differentiation of lung fibroblasts into myofibroblasts, leading to excessive deposition of collagen proteins and progressive scarring. We have previously shown that synthesis of collagen by myofibroblasts requires de novo synthesis of glycine, the most abundant amino acid found in collagen protein. TGF-β upregulates the expression of the enzymes of the de novo serine/glycine synthesis pathway in lung fibroblasts; however, the transcriptional and signaling regulators of this pathway remain incompletely understood. Here we demonstrate that TGF-β promotes accumulation of Activating Transcription Factor 4 (ATF4) which is required for increased expression of the serine/glycine synthesis pathway enzymes in response to TGF-β. We found that induction of the Integrated Stress Response (ISR) contributes to TGF-β-induced ATF4 activity; however, the primary driver of ATF4 downstream of TGF-β is activation of the Mechanistic Target of Rapamycin Complex 1 (mTORC1). TGF-β activates the PI3-kinase-Akt-mTOR pathway, and inhibition of PI3-kinase prevents activation of downstream signaling and induction of ATF4. Using a panel of mTOR inhibitors, we found that ATF4 activation is dependent on mTORC1, independent of mTORC2. Rapamycin, which incompletely and allosterically inhibits mTORC1 had no effect on TGF-β-mediated induction of ATF4; however, Rapalink-1, which specifically targets the kinase domain of mTORC1 completely inhibited ATF4 induction and metabolic reprogramming downstream of TGF-β. Our results provide insight into the mechanisms of metabolic reprogramming in myofibroblasts and clarify contradictory published findings on the role of mTOR inhibition in myofibroblast differentiation.
    Keywords:  Fibrosis; Glycolysis; Metabolism; Mitochondria
  4. Front Endocrinol (Lausanne). 2020 ;11 403
      This minireview is a brief overview examining the roles of insulin-like growth factors (IGFs) and the PI3K/Akt pathway in two apparently unconnected diseases: Alzheimer's dementia and cancer. For both, increased age is a major risk factor, and, in accord with the global rise in average life expectancy, their prevalence is also increasing. Cancer, however, involves excessive cell proliferation and metastasis, whereas Alzheimer's disease (AD) involves cell death and tissue destruction. The apparent "inverse" nature of these disease states is examined here, but also some important commonalities in terms of the PI3K/Akt pathway, glucose utilization and cell deregulation/death. The focus here is on four key molecules associated with this pathway; notably, the insulin receptor substrate 1 (IRS-1), cellular tumor antigen p53 (p53), peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1) and low-density lipoprotein receptor-related protein-1 (LRP1), all previously identified as potential therapeutic targets for both diseases. The insulin-resistant state, commonly reported in AD brain, results in neuronal glucose deprivation, due to a dampening down of the PI3K/Akt pathway, including overactivity of the mammalian target of rapamycin 1 (mTORC1) complex, hyperphosphorylation of p53 and neuronal death. This contrasts with cancer, where there is overstimulation of the PI3K/Akt pathway and the suppression of mTORC1 and p53, enabling abundant energy and unrestrained cell proliferation. Although these disease states appear to be diametrically opposed, the same key molecules are controlling pathology and, with differential targeting of therapeutics, may yet provide a beneficial outcome for both.
    Keywords:  Alzheimer's; IGF-1; LRP1; PI3K/Akt pathway; PIN1; cancer; insulin; p53
  5. EMBO Mol Med. 2020 Jul 16. e11987
      Triple-negative breast cancer (TNBC) has poorer prognosis compared to other types of breast cancers due to the lack of effective therapies and markers for patient stratification. Loss of PTEN tumor suppressor gene expression is a frequent event in TNBC, resulting in over-activation of the PI 3-kinase (PI3K) pathway and sensitivity to its inhibition. However, PI3K pathway inhibitors show limited efficacy as monotherapies on these tumors. We report a whole-genome screen to identify targets whose inhibition enhanced the effects of different PI3K pathway inhibitors on PTEN-null TNBC. This identified a signaling network that relies on both the G protein-coupled receptor for thrombin (PAR1/F2R) and downstream G protein βγ subunits and also epidermal growth factor receptor (EGFR) for the activation of the PI3K isoform p110β and AKT. Compensation mechanisms involving these two branches of the pathway could bypass PI3K blockade, but combination targeting of both EGFR and PI3Kβ suppressed ribosomal protein S6 phosphorylation and exerted anti-tumor activity both in vitro and in vivo, suggesting a new potential therapeutic strategy for PTEN-null TNBC.
    Keywords:   PTEN ; G protein; p110β; resistance; triple-negative breast cancer
  6. Cell Commun Signal. 2020 Jul 17. 18(1): 114
      BACKGROUND: Angiogenesis plays an important role in the survival of tissues, as blood vessels provide oxygen and nutrients required by the resident cells. Thus, targeting angiogenesis is a prominent strategy in many different settings, including both tissue engineering and cancer treatment. However, not all of the approaches that modulate angiogenesis lead to successful outcomes. Angiogenesis-based therapies primarily target pro-angiogenic factors such as vascular endothelial growth factor-A (VEGF) or fibroblast growth factor (FGF) in isolation, and there is a limited understanding of how these promoters combine together to stimulate angiogenesis. Targeting one pathway could be insufficient, as alternative pathways may compensate, diminishing the overall effect of the treatment strategy.METHODS: To gain mechanistic insight and identify novel therapeutic strategies, we have developed a detailed mathematical model to quantitatively characterize the crosstalk of FGF and VEGF intracellular signaling. The model focuses on FGF- and VEGF-induced mitogen-activated protein kinase (MAPK) signaling to promote cell proliferation and the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathway, which promotes cell survival and migration. We fit the model to published experimental datasets that measure phosphorylated extracellular regulated kinase (pERK) and Akt (pAkt) upon FGF or VEGF stimulation. We validate the model with separate sets of data.
    RESULTS: We apply the trained and validated mathematical model to characterize the dynamics of pERK and pAkt in response to the mono- and co-stimulation by FGF and VEGF. The model predicts that for certain ranges of ligand concentrations, the maximum pERK level is more responsive to changes in ligand concentration compared to the maximum pAkt level. Also, the combination of FGF and VEGF indicates a greater effect in increasing the maximum pERK compared to the summation of individual effects, which is not seen for maximum pAkt levels. In addition, our model identifies the influential species and kinetic parameters that specifically modulate the pERK and pAkt responses, which represent potential targets for angiogenesis-based therapies.
    CONCLUSIONS: Overall, the model predicts the combination effects of FGF and VEGF stimulation on ERK and Akt quantitatively and provides a framework to mechanistically explain experimental results and guide experimental design. Thus, this model can be utilized to study the effects of pro- and anti-angiogenic therapies that particularly target ERK and/or Akt activation upon stimulation with FGF and VEGF. Video Abstract.
    Keywords:  Angiogenesis; Computational modeling; Growth factor signaling; Sensitivity analysis
  7. J Biochem. 2020 Jul 11. pii: mvaa076. [Epub ahead of print]
      Ragulator is a heteropentameric protein complex consisting of two roadblock heterodimers wrapped by the membrane anchor p18/Lamtor1. The Ragulator complex functions as a lysosomal membrane scaffold for Rag GTPases to recruit and activate mTORC1. However, the roles of Ragulator structure in the regulation of mTORC1 function remain elusive. In this study, we disrupted Ragulator structure by directly anchoring RagC to lysosomes and monitored the effect on amino acid-dependent mTORC1 activation. Expression of lysosome-anchored RagC in p18-deficient cells resulted in constitutive lysosomal localization and amino acid-independent activation of mTORC1. Co-expression of Ragulator in this system restored the amino acid dependency of mTORC1 activation. Furthermore, ablation of Gator1, a suppressor of Rag GTPases, induced amino acid-independent activation of mTORC1 even in the presence of Ragulator. These results demonstrate that Ragulator structure is essential for amino acid-dependent regulation of Rag GTPases via Gator1. In addition, our genetic analyses revealed new roles of amino acids in the regulation of mTORC1 as follows: amino acids could activate a fraction of mTORC1 in a Rheb-independent manner, and could also drive negative-feedback regulation of mTORC1 signaling via protein phosphatases. These intriguing findings contribute to our overall understanding of the regulatory mechanisms of mTORC1 signaling.
    Keywords:  Rag; Ragulator; Rheb; lysosome; mTORC1; p18
  8. Nat Commun. 2020 Jul 13. 11(1): 3455
      CRISPR-based genetic screening has revolutionized cancer drug target discovery, yet reliable, multiplex gene editing to reveal synergies between gene targets remains a major challenge. Here, we present a simple and robust CRISPR-Cas12a-based approach for combinatorial genetic screening in cancer cells. By engineering the CRISPR-AsCas12a system with key modifications to the Cas protein and its CRISPR RNA (crRNA), we can achieve high efficiency combinatorial genetic screening. We demonstrate the performance of our optimized AsCas12a (opAsCas12a) through double knockout screening against epigenetic regulators. This screen reveals synthetic sick interactions between Brd9&Jmjd6, Kat6a&Jmjd6, and Brpf1&Jmjd6 in leukemia cells.
  9. J Cell Biol. 2020 Sep 07. pii: e201910041. [Epub ahead of print]219(9):
      The tumor suppressor PTEN is essential for early development. Its lipid phosphatase activity converts PIP3 to PIP2 and antagonizes the PI3K-Akt pathway. In this study, we demonstrate that PTEN's protein phosphatase activity is required for epiblast epithelial differentiation and polarization. This is accomplished by reconstitution of PTEN-null embryoid bodies with PTEN mutants that lack only PTEN's lipid phosphatase activity or both PTEN's lipid and protein phosphatase activities. Phosphotyrosine antibody immunoprecipitation and mass spectrometry were used to identify Abi1, a core component of the WASP-family verprolin homologous protein (WAVE) regulatory complex (WRC), as a new PTEN substrate. We demonstrate that PTEN dephosphorylation of Abi1 at Y213 and S216 results in Abi1 degradation through the calpain pathway. This leads to down-regulation of the WRC and reorganization of the actin cytoskeleton. The latter is critical to the transformation of nonpolar pluripotent stem cells into the polarized epiblast epithelium. Our findings establish a link between PTEN and WAVE-Arp2/3-regulated actin cytoskeletal dynamics in epithelial morphogenesis.
  10. Biochem Soc Trans. 2020 Jul 17. pii: BST20190845. [Epub ahead of print]
      The phosphatidylinositol 3-kinase (PI3K) pathway is a critical regulator of many cellular processes including cell survival, growth, proliferation and motility. Not surprisingly therefore, the PI3K pathway is one of the most frequently mutated pathways in human cancers. In addition to their canonical role as part of the PI3K holoenzyme, the class IA PI3K regulatory subunits undertake critical functions independent of PI3K. The PI3K regulatory subunits exist in excess over the p110 catalytic subunits and therefore free in the cell. p110-independent p85 is unstable and exists in a monomer-dimer equilibrium. Two conformations of dimeric p85 have been reported that are mediated by N-terminal and C-terminal protein domain interactions, respectively. The role of p110-independent p85 is under investigation and it has been found to perform critical adaptor functions, sequestering or influencing compartmentalisation of key signalling proteins. Free p85 has roles in glucose homeostasis, cellular stress pathways, receptor trafficking and cell migration. As a regulator of fundamental pathways, the amount of p110-independent p85 in the cell is critical. Factors that influence the monomer-dimer equilibrium of p110-independent p85 offer additional control over this system, disruption to which likely results in disease. Here we review the current knowledge of the structure and functions of p110-independent class IA PI3K regulatory subunits.
    Keywords:  cytoskeleton; nuclear functions; p85; phosphoinositide 3-kinase; receptor trafficking; regulatory dimers
  11. Future Oncol. 2020 Jul 13.
      B-cell receptor signaling is important in the pathogenesis of non-Hodgkin lymphoma. The PI3K pathway is activated by B-cell receptor signaling. Recently, several PI3K inhibitors have been in development for the treatment of indolent non-Hodgkin lymphomas. Copanlisib is a PI3Kα and PI3Kδ inhibitor that has been approved for its use as third-line therapy in the treatment of relapsed or refractory follicular lymphoma. The two other PI3k inhibitors approved by the US FDA in this setting are idelalisib and duvelisib. In this review, we compare the efficacy and adverse event profile of these different PI3K inhibitors and discuss the advantages and challenges of using copanlisib along with a guide on managing routinely encountered adverse events in the clinics.
    Keywords:  PI3K inhibitor therapy; copanlisib; lymphoma; targeted therapy
  12. ESMO Open. 2020 Jul;pii: e000672. [Epub ahead of print]5(4):
      BACKGROUND: Glioblastoma relapse is associated with activation of phosphatidylinositol 3-kinase (PI3K) signalling pathway. In preclinical studies, the pan-PI3K inhibitor buparlisib showed antitumour activity in glioma models.METHODS: This was a two-part, multicentre, phase Ib/II study in patients with recurrent glioblastoma pretreated with radiotherapy and temozolomide standard of care. Patients received buparlisib (80 mg or 100 mg once daily) plus carboplatin (area under the curve (AUC)=5 every 3 weeks), or buparlisib (60 mg once daily) plus lomustine (100 mg/m2 every 6 weeks). The primary endpoint was to determine the maximum tolerable dose (MTD) and/or recommended phase II dose of buparlisib plus carboplatin or lomustine.
    RESULTS: Between 28 February 2014 and 7 July 2016, 35 patients were enrolled and treated with buparlisib plus carboplatin (n=17; buparlisib (80 mg) plus carboplatin, n=3; and buparlisib (100 mg) plus carboplatin, n=14), or buparlisib (60 mg) plus lomustine (n=18). The MTD of buparlisib was determined to be 100 mg per day in combination with carboplatin at an AUC of 5 every 3 weeks. The MTD of buparlisib in combination with lomustine could not be determined as it did not satisfy the MTD criteria per the Bayesian logistic regression model.
    CONCLUSION: The overall safety profile of buparlisib remained unchanged, and no new or unexpected safety findings were reported in this study. Preliminary assessment for both combinations did not demonstrate sufficient antitumour activity compared with historical data on single-agent carboplatin or lomustine.
    Keywords:  BKM120; buparlisib; rGBM; recurrent glioblastoma
  13. Nat Biotechnol. 2020 Jul 13.
      Cas12a RNA-guided endonucleases are promising tools for multiplexed genetic perturbations because they can process multiple guide RNAs expressed as a single transcript, and subsequently cleave target DNA. However, their widespread adoption has lagged behind Cas9-based strategies due to low activity and the lack of a well-validated pooled screening toolkit. In the present study, we describe the optimization of enhanced Cas12a from Acidaminococcus (enAsCas12a) for pooled, combinatorial genetic screens in human cells. By assaying the activity of thousands of guides, we refine on-target design rules and develop a comprehensive set of off-target rules to predict and exclude promiscuous guides. We also identify 38 direct repeat variants that can substitute for the wild-type sequence. We validate our optimized AsCas12a toolkit by screening for synthetic lethalities in OVCAR8 and A375 cancer cells, discovering an interaction between MARCH5 and WSB2. Finally, we show that enAsCas12a delivers similar performance to Cas9 in genome-wide dropout screens but at greatly reduced library size, which will facilitate screens in challenging models.
  14. Cell Metab. 2020 Jul 09. pii: S1550-4131(20)30318-1. [Epub ahead of print]
      Rapid alterations in cellular metabolism allow tissues to maintain homeostasis during changes in energy availability. The central metabolic regulator acetyl-CoA carboxylase 2 (ACC2) is robustly phosphorylated during cellular energy stress by AMP-activated protein kinase (AMPK) to relieve its suppression of fat oxidation. While ACC2 can also be hydroxylated by prolyl hydroxylase 3 (PHD3), the physiological consequence thereof is poorly understood. We find that ACC2 phosphorylation and hydroxylation occur in an inverse fashion. ACC2 hydroxylation occurs in conditions of high energy and represses fatty acid oxidation. PHD3-null mice demonstrate loss of ACC2 hydroxylation in heart and skeletal muscle and display elevated fatty acid oxidation. Whole body or skeletal muscle-specific PHD3 loss enhances exercise capacity during an endurance exercise challenge. In sum, these data identify an unexpected link between AMPK and PHD3, and a role for PHD3 in acute exercise endurance capacity and skeletal muscle metabolism.
    Keywords:  Prolyl hydroxylase 3; acetyl-CoA carboxylase 2 modification; exercise capacity; fat catabolism
  15. Nat Biomed Eng. 2020 Jul;4(7): 686-703
      Theranostic agents should ideally be renally cleared and biodegradable. Here, we report the synthesis, characterization and theranostic applications of fluorescent ultrasmall gold quantum clusters that are stabilized by the milk metalloprotein alpha-lactalbumin. We synthesized three types of these nanoprobes that together display fluorescence across the visible and near-infrared spectra when excited at a single wavelength through optical colour coding. In live tumour-bearing mice, the near-infrared nanoprobe generates contrast for fluorescence, X-ray computed tomography and magnetic resonance imaging, and exhibits long circulation times, low accumulation in the reticuloendothelial system, sustained tumour retention, insignificant toxicity and renal clearance. An intravenously administrated near-infrared nanoprobe with a large Stokes shift facilitated the detection and image-guided resection of breast tumours in vivo using a smartphone with modified optics. Moreover, the partially unfolded structure of alpha-lactalbumin in the nanoprobe helps with the formation of an anti-cancer lipoprotein complex with oleic acid that triggers the inhibition of the MAPK and PI3K-AKT pathways, immunogenic cell death and the recruitment of infiltrating macrophages. The biodegradability and safety profile of the nanoprobes make them suitable for the systemic detection and localized treatment of cancer.
  16. Diagnostics (Basel). 2020 Jul 09. pii: E467. [Epub ahead of print]10(7):
      The PI3K/AKT/mTOR signaling pathway is significantly activated in rheumatoid arthritis. In addition, somatic activating mutations of the PI3K/AKT/mTOR pathway may result in PIK3CA-related overgrowth spectrum diseases, including CLOVES (Congenital Lipomatous Overgrowth, Vascular malformation, Epidermal nevi, Skeletal abnormalities/Scoliosis) syndrome. We describe the case of a young female patient, with anti-citrullinated peptide antibodies-positive rheumatoid arthritis, referred for persistent finger pain and stiffness. Examination revealed discrete macrodactyly involving two fingers, scoliosis, asymmetrical calves, venectasias, a shoulder nevus and triangular feet with a "sandal gap" between two toes. These mild dysmorphic features with early-onset and the history of surgeries for thoracic lipoma and venous malformation were strongly suggestive of CLOVES syndrome. Confirmatory mutation analysis was not performed, as blood or saliva testing is not contributive for tissue-specific localized effects in the PIK3CA-related overgrowth spectrum. Nevertheless, lack of detection of a PIK3CA mutation does not exclude the diagnosis in patients fulfilling clinical criteria. Due to the patient's wish to plan a pregnancy, therapy consisted in sulfasalazine and hydroxychloroquine, along with orthotic correction of leg length discrepancy. Overgrowth syndromes and arthritis may share common pathways. Mild macrodactyly should be differentiated from dactylitis. Diagnosing patients with minimal dysmorphic features within the PI3K-related overgrowth spectrum may help design better care strategies, in the quest for personalized medicine.
    Keywords:  CLOVES syndrome; PIK3/AKT/mTOR pathway; PIK3CA-related overgrowth spectrum; macrodactyly; rheumatoid arthritis
  17. Nat Commun. 2020 Jul 14. 11(1): 3520
      PRDM (PRDI-BF1 and RIZ homology domain containing) family members are sequence-specific transcriptional regulators involved in cell identity and fate determination, often dysregulated in cancer. The PRDM15 gene is of particular interest, given its low expression in adult tissues and its overexpression in B-cell lymphomas. Despite its well characterized role in stem cell biology and during early development, the role of PRDM15 in cancer remains obscure. Herein, we demonstrate that while PRDM15 is largely dispensable for mouse adult somatic cell homeostasis in vivo, it plays a critical role in B-cell lymphomagenesis. Mechanistically, PRDM15 regulates a transcriptional program that sustains the activity of the PI3K/AKT/mTOR pathway and glycolysis in B-cell lymphomas. Abrogation of PRDM15 induces a metabolic crisis and selective death of lymphoma cells. Collectively, our data demonstrate that PRDM15 fuels the metabolic requirement of B-cell lymphomas and validate it as an attractive and previously unrecognized target in oncology.
  18. Cancer Res. 2020 Jul 14. pii: canres.1564.2019. [Epub ahead of print]
      In solid tumors, vascular structure and function varies from the core to the periphery. This structural heterogeneity has been proposed to influence the mechanisms by which tumor cells enter the circulation. Blood vessels exhibit regional defects in endothelial coverage, which can result in cancer cells directly exposed to flow and potentially promoting intravasation. Consistent with prior reports, we observed in human breast tumors and in a mouse model of breast cancer that approximately 6% of vessels consisted of both endothelial cells and tumor cells, so-called mosaic vessels. Due in part to the challenges associated with observing tumor-vessel interactions deep within tumors in real-time, the mechanisms by which mosaic vessels form remain incompletely understood. We developed a tissue-engineered model containing a physiologically realistic microvessel in co-culture with mammary tumor organoids. This approach allows real-time and quantitative assessment of tumor-vessel interactions under conditions that recapitulate many in vivo features. Imaging revealed that tumor organoids integrate into the endothelial cell lining, resulting in mosaic vessels with gaps in the basement membrane. While mosaic vessel formation was the most frequently observed interaction, tumor organoids also actively constricted and displaced vessels. Furthermore, intravasation of cancer cell clusters was observed following the formation of a mosaic vessel. Taken together, our data reveal that cancer cells can rapidly reshape, destroy, or integrate into existing blood vessels, thereby affecting oxygenation, perfusion, and systemic dissemination. Our novel assay also enables future studies to identify targetable mechanisms of vascular recruitment and intravasation.
  19. J Cutan Pathol. 2020 Jul 14.
      BACKGROUND: Angiolipoma (AL) is considered as a lipoma variant, characterized by the combination of mature adipocytes and capillary blood vessels diffusely distributed within the tumor. With the exception of recurrent PRKD2 mutations of uncertain pathogenetic significance, the genetic abnormalities of ALs are unknown, in absence of any of the specific chromosomal aberrations described in other lipoma variants.METHODS: Formalin-fixed and paraffin-embedded blocks of 13 conventional ALs and 5 cellular ALs from 17 individuals were retrieved and analyzed for mutations in exons 9 and 20 of PIK3CA by polymerase chain reaction and Sanger sequencing.
    RESULTS: Activating PIK3CA mutations were identified in 14 tumors (78%). All PIK3CA-mutated samples carried the same exon 9 mutation, c.1634A>C (p.E545A). No mutation was detected in exon 20 of PIK3CA. No significant difference between PIK3CA-mutated and wild-type samples appeared to exist based on age, gender, and location of the tumor. All 5 cellular ALs carried the p.E545A PIK3CA mutation.
    CONCLUSIONS: The high frequency of the p.E545A PIK3CA mutation in both conventional and cellular ALs suggests that activation of the PI3K/AKT pathway plays a key role in AL pathogenesis, and reinforces the concept that cellular AL should be regarded as a variant of AL. This article is protected by copyright. All rights reserved.
    Keywords:  Angiolipoma; PIK3CA; hemangioma; lipoma; vascular malformation
  20. ESMO Open. 2020 Jul;pii: e000673. [Epub ahead of print]5(4):
      BACKGROUND: Most glioblastoma tumours exhibit intrinsic phosphatidylinositol 3-kinase (PI3K) pathway activation. Preclinical in vitro and in vivo models suggest that buparlisib (an oral pan-PI3K inhibitor) can have an effect on glioblastoma directly and by enhancing the activity of radiation and of temozolomide.METHODS: This was a phase I, two-stage, multicentre, open-label, dose-escalation study of buparlisib in combination with temozolomide and radiotherapy in patients with newly diagnosed glioblastoma. In stage I, patients who completed the concomitant phase of combination of temozolomide and radiation prior to study entry received buparlisib in combination with temozolomide. In stage II, patients received buparlisib in combination with temozolomide and radiotherapy in the concomitant phase and temozolomide in the adjuvant treatment phase. The primary objective was to estimate the maximum tolerated dose (MTD) of buparlisib when combined with the approved first-line treatment of temozolomide and radiotherapy.
    RESULTS: The MTD of buparlisib in combination with temozolomide at stage I (adjuvant phase only) was 80 mg/day, which was used as the starting dose in stage II. The MTD of buparlisib in combination with temozolomide and radiotherapy in stage II (concomitant + adjuvant phase) was not determined due to the observed dose-limiting toxicities and treatment discontinuations due to adverse events (AEs). In stage I, the most commonly reported AEs were nausea (72.7%) and fatigue (59.1%). In stage II, the most commonly reported AEs were fatigue and nausea (56.3% each). No on-treatment deaths were reported during the study.
    CONCLUSION: Considering that the primary objective of estimating the MTD was not achieved in addition to the observed challenging safety profile of buparlisib in combination with radiotherapy and temozolomide, Novartis decided not to pursue the development of buparlisib in newly diagnosed glioblastoma.Trial registration identifier: NCT01473901.
    Keywords:  BKM120; buparlisib; glioblastoma
  21. Nat Commun. 2020 Jul 17. 11(1): 3616
      Genomic and precision medicine research has afforded notable advances in human cancer treatment, yet applicability to other species remains uncertain. Through whole-exome and transcriptome analyses of 191 spontaneous canine mammary tumors (CMTs) that exhibit the archetypal features of human breast cancers, we found a striking resemblance of genomic characteristics including frequent PIK3CA mutations (43.1%), aberrations of the PI3K-Akt pathway (61.7%), and key genes involved in cancer initiation and progression. We also identified three gene expression-based CMT subtypes, one of which segregated with basal-like human breast cancer subtypes with activated epithelial-to-mesenchymal transition, low claudin expression, and unfavorable disease prognosis. A relative lack of ERBB2 amplification and Her2-enrichment subtype in CMT denoted species-specific molecular mechanisms. Taken together, our results elucidate cross-species oncogenic signatures for a better understanding of universal and context-dependent mechanisms in breast cancer development and provide a basis for precision diagnostics and therapeutics for domestic dogs.
  22. Bioinformatics. 2020 Jul 11. pii: btaa590. [Epub ahead of print]
      MOTIVATION: Misregulation of signaling pathway activity is etiologic for many human diseases, and modulating activity of signaling pathways is often the preferred therapeutic strategy. Understanding the mechanism of action (MOA) of bioactive chemicals in terms of targeted signaling pathways is the essential first step in evaluating their therapeutic potential. Changes in signaling pathway activity are often not reflected in changes in expression of pathway genes which makes MOA inferences from transcriptional signatures a difficult problem.RESULTS: We developed a new computational method for implicating pathway targets of bioactive chemicals and other cellular perturbations by integrated analysis of pathway network topology, the LINCS transcriptional signatures of genetic perturbations of pathway genes and the transcriptional signature of the perturbation. Our methodology accurately predicts signaling pathways targeted by the perturbation when current pathway analysis approaches utilizing only a transcriptional signature of the perturbation fail.
    AVAILABILITY: Open source R package paslincs is available at
    SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
  23. Proc Natl Acad Sci U S A. 2020 Jul 13. pii: 202000362. [Epub ahead of print]
      Spermatogonial stem cells (SSCs) are essential for the generation of sperm and have potential therapeutic value for treating male infertility, which afflicts >100 million men world-wide. While much has been learned about rodent SSCs, human SSCs remain poorly understood. Here, we molecularly characterize human SSCs and define conditions favoring their culture. To achieve this, we first identified a cell-surface protein, PLPPR3, that allowed purification of human primitive undifferentiated spermatogonia (uSPG) highly enriched for SSCs. Comparative RNA-sequencing analysis of these enriched SSCs with differentiating SPG (KIT+ cells) revealed the full complement of genes that shift expression during this developmental transition, including genes encoding key components in the TGF-β, GDNF, AKT, and JAK-STAT signaling pathways. We examined the effect of manipulating these signaling pathways on cultured human SPG using both conventional approaches and single-cell RNA-sequencing analysis. This revealed that GDNF and BMP8B broadly support human SPG culture, while activin A selectively supports more advanced human SPG. One condition-AKT pathway inhibition-had the unique ability to selectively support the culture of primitive human uSPG. This raises the possibility that supplementation with an AKT inhibitor could be used to culture human SSCs in vitro for therapeutic applications.
    Keywords:  PLPPR3; RNA sequencing; human spermatogonia; in vitro culture; testis
  24. Int J Mol Sci. 2020 Jul 11. pii: E4902. [Epub ahead of print]21(14):
      Stemness and reprogramming involve transcriptional master regulators that suppress cell differentiation while promoting self-renewal. A distinguished example thereof is SOX2, a high mobility group (HMG)-box transcription factor (TF), whose subcellular localization and turnover regulation in embryonic, induced-pluripotent, and cancer stem cells (ESCs, iPSCs, and CSCs, respectively) is mediated by the PI3K/AKT/SOX2 axis, a stem cell-specific branch of the PI3K/AKT signaling pathway. Further effector functions associated with PI3K/AKT induction include cell cycle progression, cellular (mass) growth, and the suppression of apoptosis. Apoptosis, however, is a central element of DNA damage response (DDR), where it provides a default mechanism for cell clearance when DNA integrity cannot be maintained. A key player in DDR is tumor suppressor p53, which accumulates upon DNA-damage and is counter-balanced by PI3K/AKT enforced turnover. Accordingly, stemness sustaining SOX2 expression and p53-dependent DDR mechanisms show molecular-functional overlap in PI3K/AKT signaling. This constellation proves challenging for stem cells whose genomic integrity is a functional imperative for normative ontogenesis. Unresolved mutations in stem and early progenitor cells may in fact provoke transformation and cancer development. Such mechanisms are also particularly relevant for iPSCs, where genetic changes imposed through somatic cell reprogramming may promote DNA damage. The current review aims to summarize the latest advances in the understanding of PI3K/AKT/SOX2-driven stemness and its intertwined relations to p53-signaling in DDR under conditions of pluripotency, reprogramming, and transformation.
    Keywords:  DNA damage response (DDR); DNA maintenance; PI3K/AKT signaling; SOX2; cancer; genome integrity; p53; pluripotency; stem cells; transformation
  25. Development. 2020 Jul 15. pii: dev181495. [Epub ahead of print]147(14):
      During development, cells need to make decisions about their fate in order to ensure that the correct numbers and types of cells are established at the correct time and place in the embryo. Such cell fate decisions are often classified as deterministic or stochastic. However, although these terms are clearly defined in a mathematical sense, they are sometimes used ambiguously in biological contexts. Here, we provide some suggestions on how to clarify the definitions and usage of the terms stochastic and deterministic in biological experiments. We discuss the frameworks within which such clear definitions make sense and highlight when certain ambiguity prevails. As an example, we examine how these terms are used in studies of neuronal cell fate decisions and point out areas in which definitions and interpretations have changed and matured over time. We hope that this Review will provide some clarification and inspire discussion on the use of terminology in relation to fate decisions.
    Keywords:  Central nervous system; Fate decisions; Stochasticity
  26. Nature. 2020 Jul 15.
      Ageing is the single greatest cause of disease and death worldwide, and understanding the associated processes could vastly improve quality of life. Although major categories of ageing damage have been identified-such as altered intercellular communication, loss of proteostasis and eroded mitochondrial function1-these deleterious processes interact with extraordinary complexity within and between organs, and a comprehensive, whole-organism analysis of ageing dynamics has been lacking. Here we performed bulk RNA sequencing of 17 organs and plasma proteomics at 10 ages across the lifespan of Mus musculus, and integrated these findings with data from the accompanying Tabula Muris Senis2-or 'Mouse Ageing Cell Atlas'-which follows on from the original Tabula Muris3. We reveal linear and nonlinear shifts in gene expression during ageing, with the associated genes clustered in consistent trajectory groups with coherent biological functions-including extracellular matrix regulation, unfolded protein binding, mitochondrial function, and inflammatory and immune response. Notably, these gene sets show similar expression across tissues, differing only in the amplitude and the age of onset of expression. Widespread activation of immune cells is especially pronounced, and is first detectable in white adipose depots during middle age. Single-cell RNA sequencing confirms the accumulation of T cells and B cells in adipose tissue-including plasma cells that express immunoglobulin J-which also accrue concurrently across diverse organs. Finally, we show how gene expression shifts in distinct tissues are highly correlated with corresponding protein levels in plasma, thus potentially contributing to the ageing of the systemic circulation. Together, these data demonstrate a similar yet asynchronous inter- and intra-organ progression of ageing, providing a foundation from which to track systemic sources of declining health at old age.
  27. Front Cell Dev Biol. 2020 ;8 510
      Luminal pH and the distinctive distribution of phosphatidylinositol phosphate (PIP) lipids are central identifying features of organelles in all eukaryotic cells that are also critical for organelle function. V-ATPases are conserved proton pumps that populate and acidify multiple organelles of the secretory and the endocytic pathway. Complete loss of V-ATPase activity causes embryonic lethality in higher animals and conditional lethality in yeast, while partial loss of V-ATPase function is associated with multiple disease states. On the other hand, many cancer cells increase their virulence by upregulating V-ATPase expression and activity. The pH of individual organelles is tightly controlled and essential for function, but the mechanisms for compartment-specific pH regulation are not completely understood. There is substantial evidence indicating that the PIP content of membranes influences organelle pH. We present recent evidence that PIPs interact directly with subunit isoforms of the V-ATPase to dictate localization of V-ATPase subpopulations and participate in their regulation. In yeast cells, which have only one set of organelle-specific V-ATPase subunit isoforms, the Golgi-enriched lipid PI(4)P binds to the cytosolic domain of the Golgi-enriched a-subunit isoform Stv1, and loss of PI(4)P binding results in mislocalization of Stv1-containing V-ATPases from the Golgi to the vacuole/lysosome. In contrast, levels of the vacuole/lysosome-enriched signaling lipid PI(3,5)P2 affect assembly and activity of V-ATPases containing the Vph1 a-subunit isoform. Mutations in the Vph1 isoform that disrupt the lipid interaction increase sensitivity to stress. These studies have decoded "zip codes" for PIP lipids in the cytosolic N-terminal domain of the a-subunit isoforms of the yeast V-ATPase, and similar interactions between PIP lipids and the V-ATPase subunit isoforms are emerging in higher eukaryotes. In addition to direct effects on the V-ATPase, PIP lipids are also likely to affect organelle pH indirectly, through interactions with other membrane transporters. We discuss direct and indirect effects of PIP lipids on organelle pH, and the functional consequences of the interplay between PIP lipid content and organelle pH.
    Keywords:  Golgi apparatus; PIKfyve; V-ATPase; acidification; endosome; lysosome; organelle; phosphatidylinositol phosphate
  28. Prostate Cancer Prostatic Dis. 2020 Jul 13.
      BACKGROUND: The loss of PTEN function presents in up to 50% of late-stage prostate cancers, and is therefore a potential target for therapeutics. PTEN-deficient cells depend on de novo pyrimidine synthesis, a feature that can present a vulnerability.METHODS: We utilized in vitro growth assays and in vivo xenograft models to test the effect of de novo pyrimidine synthesis inhibition on prostate cell lines.
    RESULTS: Here, we demonstrate that PTEN-deficient prostate cancer cell lines are susceptible to inhibition of de novo pyrimidine synthesis by leflunomide. Tumor growth inhibition was observed in vitro and in vivo following leflunomide treatment, and is likely due to an overwhelming accumulation of DNA damage.
    CONCLUSIONS: Our work highlights that synthetic lethality arises upon the combination of PTEN loss and leflunomide treatment in prostate cancer, and may present a therapeutic opportunity for this patient population.
  29. Nature. 2020 Jul 15.
      Ageing is characterized by a progressive loss of physiological integrity, leading to impaired function and increased vulnerability to death1. Despite rapid advances over recent years, many of the molecular and cellular processes that underlie the progressive loss of healthy physiology are poorly understood2. To gain a better insight into these processes, here we generate a single-cell transcriptomic atlas across the lifespan of Mus musculus that includes data from 23 tissues and organs. We found cell-specific changes occurring across multiple cell types and organs, as well as age-related changes in the cellular composition of different organs. Using single-cell transcriptomic data, we assessed cell-type-specific manifestations of different hallmarks of ageing-such as senescence3, genomic instability4 and changes in the immune system2. This transcriptomic atlas-which we denote Tabula Muris Senis, or 'Mouse Ageing Cell Atlas'-provides molecular information about how the most important hallmarks of ageing are reflected in a broad range of tissues and cell types.
  30. Front Mol Biosci. 2020 ;7 117
      During translation elongation, the ribosome serially adds amino acids to a growing polypeptide over many rounds of catalysis. The ribosome remains bound to mRNAs over these multiple catalytic cycles, requiring high processivity. Despite its importance to translation, relatively little is known about how mRNA sequences or signaling pathways might enhance or reduce ribosome processivity. Here, we describe a metric for ribosome processivity, the ribosome density index (RDI), which is readily calculated from ribosomal profiling data. We show that ribosome processivity is not strongly influenced by open-reading frame (ORF) length or codon optimality. However, we do observe that ribosome processivity exists in two phases and that the early phase of ribosome processivity is enhanced by mTORC1, a key translational regulator. By showing that ribosome processivity is regulated, our findings suggest an additional layer of control that the cell can exert to govern gene expression.
    Keywords:  mTOR; ribosome density index; ribosome processivity; ribosome profiling; translation elongation
  31. Genet Med. 2020 Jul 14.
      Mosaicism denotes an individual who has at least two populations of cells with distinct genotypes that are derived from a single fertilized egg. Genetic variation among the cell lines can involve whole chromosomes, structural or copy-number variants, small or single-nucleotide variants, or epigenetic variants. The mutational events that underlie mosaic variants occur during mitotic cell divisions after fertilization and zygote formation. The initiating mutational event can occur in any types of cell at any time in development, leading to enormous variation in the distribution and phenotypic effect of mosaicism. A number of classification proposals have been put forward to classify genetic mosaicism into categories based on the location, pattern, and mechanisms of the disease. We here propose a new classification of genetic mosaicism that considers the affected tissue, the pattern and distribution of the mosaicism, the pathogenicity of the variant, the direction of the change (benign to pathogenic vs. pathogenic to benign), and the postzygotic mutational mechanism. The accurate and comprehensive categorization and subtyping of mosaicisms is important and has potential clinical utility to define the natural history of these disorders, tailor follow-up frequency and interventions, estimate recurrence risks, and guide therapeutic decisions.
    Keywords:  mosaicism; mutational event; new classification; postzygotic
  32. Oncotarget. 2020 Jun 30. 11(26): 2493-2511
      Triple negative breast cancer (TNBC) is a heterogenous and lethal disease that lacks diagnostic markers and therapeutic targets; as such common targets are highly sought after. IQGAP1 is a signaling scaffold implicated in TNBC, but its mechanism is unknown. Here we show that IQGAP1 localizes to the centrosome, interacts with and influences the expression level and localization of key centrosome proteins like BRCA1 and thereby impacts centrosome number. Genetic mutant analyses suggest that phosphorylation cycling of IQGAP1 is important to its subcellular localization and centrosome-nuclear shuttling of BRCA1; dysfunction of this process defines two alternate mechanisms associated with cell proliferation. TNBC cell lines and patient tumor tissues differentially phenocopy these mechanisms supporting clinical existence of molecularly distinct variants of TNBC defined by IQGAP1 pathways. These variants are defined, at least in part, by differential mis-localization or stabilization of IQGAP1-BRCA1 and rewiring of a novel Erk1/2-MNK1-JNK-Akt-β-catenin signaling signature. We discuss a model in which IQGAP1 modulates centrosome-nuclear crosstalk to regulate cell division and imparts on cancer. These findings have implications on cancer racial disparities and can provide molecular tools for classification of TNBC, presenting IQGAP1 as a common target amenable to personalized medicine.
    Keywords:  BRCA1; IQGAP1; MNK1; triple negative breast cancer; β-catenin
  33. Am J Physiol Endocrinol Metab. 2020 Jul 14.
      Insulin resistance in obesity and type 2 diabetes (T2D) has been shown to be associated with decreased de novo fatty acid (FA) synthesis in adipose tissue. It is known that insulin can acutely stimulate FA synthesis in adipocytes, however the mechanisms underlying this effect are unclear. The rate-limiting step in FA synthesis is catalyzed by acetyl-CoA carboxylase (ACC), known to be regulated through inhibitory phosphorylation at S79 by the AMP-activated protein kinase (AMPK). Previous results from our laboratory showed an inhibition of AMPK activity by insulin, which was accompanied by PKB-dependent phosphorylation of AMPK at S485. However, if the S485 phosphorylation is required for insulin-induced inhibition of AMPK, or if other mechanisms underlie the reduced kinase activity, is not known. In order to investigate this, primary rat adipocytes were transduced with a recombinant adenovirus encoding AMPK-WT or a non-phosphorylatable AMPK S485A mutant. AMPK activity measurements by western blotting and in vitro kinase assay revealed that WT and S485A AMPK were inhibited to a similar degree by insulin, indicating that AMPK S485 phosphorylation is not required for insulin-induced AMPK inhibition. Further analysis suggested an involvement of decreased AMP/ATP ratios in the insulin-induced inhibition of AMPK activity, whereas a possible contribution of phosphodiesterases was excluded. Furthermore, we show that insulin-induced AMPK S485 phosphorylation also occurs in human adipocytes, suggesting it to be of an importance yet to be revealed. Altogether, this study increases our understanding of how insulin regulates AMPK activity, and with that, FA synthesis, in adipose tissue.
    Keywords:  AMP-activated protein kinase; S485; adipocytes; fatty acid synthesis; insulin
  34. Gynecol Oncol. 2020 Jul 09. pii: S0090-8258(20)32332-5. [Epub ahead of print]
      PURPOSE: This study aimed to describe the prognostic value of PI3K/AKT pathway mutations in a large cohort of patients with cervical cancer.EXPERIMENTAL DESIGN: Patients with pre-treatment archival specimens, diagnosed with FIGO stages IB-IVA cervical cancer between 1998 and 2014 and treated with radical, curative intent chemoradiotherapy (CRT) at a single center were identified. Mutational status was determined by next generation sequencing and PIK3CA copy number (CNV) was assessed by digital PCR.
    RESULTS: 190 patients with available pre-treatment tumor specimens were identified. Median OS and PFS were 57.4 and 46.0 months, respectively. A total of 161 tumors were successfully sequenced; 60 (37.3%) had PI3K/AKT pathway mutations, with 50 (30.1%) having PIK3CA hotspot mutations. PIK3CA CNV gain was noted in 79 (59.2%) of the 154 successfully analyzed. On univariate analysis, PIK3CA mutation was associated with poor OS (HR 1.73; 95% CI: 1.03-2.92; p = .037) but not PFS (HR 1.38; 0.84-2.28; p = .204). Absence of any PI3K/AKT pathway mutation was associated with improved OS (HR 1.68; 1.01-2.81; p = .046) but not PFS (HR 1.50; 0.93-2.43; p = .202). Associations were not maintained when adjusting for clinical factors. On univariate analysis, PIK3CA mutation positive, CNV normal tumors were associated with poorer OS (HR 2.55; 1.18-5.50; p = .017) and trend to worse PFS (HR 1.87; 0.90-3.83; p = .094) when compared to those with CNV gain and wildtype PIK3CA.
    CONCLUSIONS: PI3K/AKT pathway mutations are common in cervical cancer. Consideration of PIK3CA mutational status with CNV status may be important in predicting outcome in cervical cancer patients.
    Keywords:  AKT; Cervical cancer; MTOR; PI3K; PIK3CA